Establishment of the human placenta (placentation) during the first trimester of pregnancy is critically dependent on cytotrophoblast (CTB) differentiation. Two pathways exist, giving rise to classes of trophoblasts with very different functions. In one pathway, CTB remain in the fetal compartment and fuse to form the syncytium that covers the chorionic villi and performs nutrient and gas exchange for the embryo. In the other pathway, which is the focus of this grant, CTB aggregate into cell columns, leave the fetal compartment and invade the uterine lining and maternal blood vessels. These columns anchor the fetus to the uterine wall. This remarkable invasive behavior is highly unusual when considered as a feature of normal cellular function. In contrast, such behavior is required for tumor metastasis. This pathway of CTB differentiation encompasses a broad spectrum of cell-cell and cell-matrix interactions and provides an outstanding opportunity to identify functionally relevant molecules by virtue of their regulated expression at transition points in the invasion process. Our hypothesis continues to be that the modulation of adhesion and matrix molecule expression, like the regulated expression of certain proteinases, is fundamental to the success of CTB invasion. Results obtained during the present grant period have documented dramatic and frequent transitions in the expression of adhesion receptors and matrix components by CTB in regions of the placenta where distinct phases of the invasion process are taking place. These results extend the histological evidence for multiple subpopulations of CTB within the first trimester placenta, by suggesting that these populations are defined by their distinctive repertoires of adhesion and extracellular matrix (ECM) molecules. This complexity, in turn, suggests that tight regulation of adhesion receptor expression is important to the overall process of CTB differentiation and invasion. To begin to address the functional role of adhesion molecules in CTB invasion, as well as the regulation of their expression, we have established cell culture systems in which first trimester CTB in vitro mimic many aspects of the invasive behavior and adhesion receptor expression exhibited in vivo. In the next funding period, we plan to study the contribution of ECM components and adhesion receptors to CTB differentiation as well as the role of growth factors in the autocrine regulation of CTB differentiation. Specifically, we will: 1) test the hypothesis that CTB-laminin interactions and CTB-fibronectin interactions play important, but distinctive roles in the stepwise process of differentiation of CTB leading to an invasive phenotype; 2) determine the role of cell-cell adhesion interaction in CTB differentiation (these studies will include direct testing of the role of E-cadherin in CTB aggregation and the regulation of invasion); and 3) localize growth regulatory factors known to be produced by first trimester CTB, and initiate studies to determine their roles in the autocrine regulation of CTB adhesion molecule expression and differentiation. Understanding the regulation of CTB invasion is critical to an understanding of the etiology of trophoblast-related disorders such as preeclampsia. In addition, it is likely that tumor cell invasion shares at least some of the mechanisms used by embryonic trophoblasts to carry out invasion.